“Canola” generally refers to plants of Brassica species that have less than 2% erucic acid (Δ13-22:1) by weight in seed oil and less than 30 micromoles of glucosinolates per gram in meal. Typically, canola oil may contain less than about 7% total saturated fatty acids and greater than 60% oleic acid (as percentages of total fatty acids). Traditionally canola crops include Brassica napus and Brassica rapa. Recently, canola quality Brassica juncea, which has oil and meal qualities similar to other canola types, has been added to the canola crop family (U.S. Pat. No. 6,303,849, to Potts et al., issued on Oct. 16, 2001; U.S. Patent Publication No. 20030221217 of 27 Nov. 2003, Yao et al; Potts and Males, 1999; all of which are incorporated herein by reference).
Fatty acid compositions of vegetable oil affect the oil quality and stability. For example, oleic acid has been recognised to have health benefits including effectiveness in lowering plasma cholesterol levels and therefore, higher levels of oleic acid content in seed oil is a desirable trait. Further, not all fatty acids in vegetable oils are equally vulnerable to high temperature and oxidation. Rather, the susceptibility of individual fatty acids to oxidation is dependent on their degree of unsaturation. For example, linolenic acid, which has three carbon-carbon double bonds, is much more vulnerable to oxidation than oleic acid that has only one carbon-carbon double bond. High oleic acid content vegetable oil is also preferred because of its heat stability. For these reasons, high oleic acid and low linolenic acid may be desirable traits in plant oils.
Plants synthesize fatty acids in their plastid as palmitoyl-ACP (16:0-ACP) and stearoyl-ACP. The conversion of stearoyl-ACP to oleoyl-ACP (18:1-ACP) is catalyzed by a soluble enzyme, the stearoyl-ACP Δ9 desaturase (Shaklin and Somerville, 1991). These acyl-ACPs are either used for glycolipid synthesis in chloroplast or transported out of chloroplast into cytoplasm as acyl-CoAs. Further desaturation of oleic acid occurs only after it is used in the synthesis of glycerolipids and incorporated into membranes, which leads to the synthesis of polyunsaturated fatty acids. The synthesis of polyunsaturated fatty acids linoleate (Δ9,12-18:2) and α-linolenate (Δ9,12,15-18:3) begins with the conversion of oleic acid (Δ9-18:1) to linoleic acid, the enzymatic step catalyzed by the microsomal ω-6 oleic acid desaturase (FAD2). The linoleic acid is then converted to α-linolenic acid through further desaturation by ω-3 linoleic acid desaturase (FAD3). There are reports that manipulation of the FAD2 gene through genetic engineering could alter fatty acid profiles. For example, heterologous expression of a soybean FAD2 gene in an Arabidopsis mutant line led to dramatic increase in the accumulation of polyunsaturated fatty acids (Heppard et al., 1996). In contrast, in a Arabidopsis mutant line fad2-5, where the transcription of FAD2 gene was decreased significantly due to T-DNA insertion, showed a dramatic increase in the accumulation of oleic acid and a significant decrease in the levels of linoleic acid and linolenic acid (Okuley et al., 1994). These findings suggest that the FAD2 gene plays an important role in controlling conversion of oleic acid to linoleic acid in seed storage lipids.
Significant efforts have been made to manipulate the fatty acid profile of plants, particularly oil-seed varieties such as Brassica spp. that are used for the large-scale production of commercial fats and oils (see for example U.S. Pat. No. 5,625,130 issued 29 Apr. 1997; U.S. Pat. No. 5,668,299 issued 16 Sep. 1997; U.S. Pat. No. 5,767,338 issued 16 Jun. 1998; U.S. Pat. No. 5,840,946 issued 24 Nov. 1998; U.S. Pat. No. 5,850,026 issued 15 Dec. 1998; U.S. Pat. No. 5,861,187 issued 19 Jan. 1999; U.S. Pat. No. 6,063,947 issued 16 May 2000; U.S. Pat. No. 6,084,157 issued 4 Jul. 2000; U.S. Pat. No. 6,169,190 issued 2 Jan. 2001; U.S. Pat. No. 6,323,392 issued 27 Nov. 2001; and international patent applications WO 97/43907 published 27 Nov. 1997 and WO 00/51415 published 8 Sep. 2000).
Brassica juncea (AB genome) is an amphidiploid plant of the Brassica genus that is generally thought to have resulted from the hybridization of Brassica rapa (A genome) and Brassica nigra (B genome). Brassica napus (AC genome) is also an amphidiploid plant of the Brassica genera but is thought to have resulted from hybridization of Brassica rapa and Brassica oleracea (C genome). Under some growing conditions, B. juncea may have certain superior traits to B. napus. These superior traits may include higher yield, better drought and heat tolerance and better disease resistance. Intensive breeding efforts have produced plants of Brassica species whose seed oil contains less than 2% erucic acid and whose de-fatted meal contains less than 30 micro moles glucosinolates per gram. The term “canola” has been used to describe varieties of Brassica spp. containing low erucic acid (Δ13-22:1) and low glucosinolates. Typically, canola oil may contain less than about 7% total saturated fatty acids and greater than 60% oleic acid (as percentages of total fatty acids). For example, in the U.S., under 21 CFR 184.1555, low erucic acid rapeseed oil derived from Brassica napus or Brassica campestris is recognized as canola oil where it has an erucic acid content of no more than 2% of the component fatty acids (Table I sets out the Food Chemicals Codex (1996) specifications for canola oil). Plant breeders have also selected canola varieties that are low in glucosinolates, such as 3-butenyl, 4-pentenyl, 2-hydroxy-3-butenyl or 2-hydroxy-4-pentenyl glucosinolate. Canola quality meal may for example be defined as having a glucosinolate content of less than 30 micromoles of aliphatic glucosinolates per gram of oil-free meal. Currently, the principle commercial canola crops comprise B. napus and B. rapa (campestris) varieties. U.S. Pat. No. 6,303,849 issued to Potts et al. on 16 Oct. 2001 (incorporated herein by reference) discloses B. juncea lines having an edible oil that has properties similar to canola. The B. juncea lines disclosed therein have a lineage that includes B. juncea lines J90-3450 and J90-4316, deposited as ATCC Accession Nos 203389 and 203390 respectively (both of which were deposited by Agriculture and Agri-Food Canada under the terms of the Budapest Treaty on 23 Oct. 1998 at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. USA 20110-2209).
TABLE AFood Chemicals Codex (1996) Specifications for Canola OilPropertyFatty Acids, % by weightCanola Oil<14<0.114:0 myristic<0.216:0 palmitic<6.016:1<1.018:0<2.518:1 oleic>50.018:2 linoleic<40.018:3 linolenic<14.020:0<1.020:1<2.022:0<0.522:1 erucic<2.024:0<0.224:1<0.2Acid value<6Cold TestPasses testColour(AOCS-Wesson)≦1.5R/15YFree fatty acids (as oleic)<0.05%Heavy metals (as Pb)≦5 mg/kgIodine value110-126Lead<0.1 mg/kgPeroxide value≦10 meq/kgRefractive index1.465-1.467Saponifiable value178-193Stability≧7 hSulfur≦10 mg/kgUnsaponifiable matter ≦1.5%Water ≦0.1%